Probing the role of single cell mechanics in disease with atomic force microscopy and microfluidics.

摘要

In the past decade, growing evidence has suggested that mechanical changes at the single cell level are correlated with disease. This dissertation focuses on technology development to quantify single cell mechanics and applications of these techniques to understand clinically relevant disease conditions in acute leukemia. The new methods are also used to test models of fundamental cell mechanical properties.In blood diseases, changes in cell mechanical properties can significantly affect the cells' ability to flow through the microvasculature. To quantify changes in deformability of blood cells and other non-adherent cells, an atomic force microscopy (AFM)-based technique was developed and characterized. Using this technique, differences in cell deformability between two different leukemia cell types were observed. This technique was then applied to investigate the role of cell mechanics in leukostasis, a poorly understood clinical condition in acute leukemia marked by leukemia cells sludging the microvasculature of vital organs.Chemotherapy, currently used as a treatment for leukemia, may actually increase risk of leukostasis---deformability measurements on leukemia patient sample cells exposed to chemotherapy revealed a two-fold order of magnitude increase in cell stiffness compared to cells not exposed to chemotherapy. This effect was avoided by disruption of the cytoskeleton.AFM was then used to measure the stiffness of individual leukemia cells taken from the peripheral blood of pediatric patients with acute lymphoblastic leukemia. The median leukemia cell stiffness was measured to be over five times higher in leukostasis symptomatic patients than in leukostasis asymptomatic patients. These results suggest that increased leukemia cell stiffness may be an additional leukostasis risk factor and should be considered in future clinical studies.To bring single cell deformability measurements towards more clinical use, a high-throughput microfluidic device was developed to quantify cell deformation through small capillary-like microchannels, a technique referred to as biophysical flow cytometry. Cell transit time and occlusion of microchannels increased in leukostasis symptomatic patient sample cells when compared to leukostasis asymptomatic patient sample cells and control neutrophil cells. While median transit time between all leukemia patient samples was consistent, the upper 25th percentile varied significantly, showing population heterogeneity may be more important than average population measurements in predicting microvasculature-related complications associated with blood diseases.A basic question underlying all mechanical measurements of cells is what constitutive model to use to interpret experimental results. Fundamental cellular mechanical properties were investigated by measuring stress propagation within adherent cells. Using AFM and high resolution three-dimensional multi-particle tracking, slow stress propagation across the cell on the order of seconds was observed. This behavior is consistent with predictions from poroelastic descriptions of the cell and has important implications for the coordination of cellular processes in response to external mechanical cues from the microenvironment.Quantification of single cell mechanical properties has proven to be a powerful tool in better understanding the pathophysiology of disease and offers new insights into the fundamental mechanical behavior of cells.